1. The Field of the Invention
The present invention relates to methods, compositions, and assays for the determination of chemical components in biological samples. More particularly, the present invention relates to calorimetric assays based on the sodium activation of amylase.
2. The Relevant Technology
Chloride ion is the major extracellular negative ion in the human body. Its main function is to maintain electrical neutrality by acting as a counter-ion to sodium. Accordingly, chloride ion levels often accompany sodium losses and excesses. Chloride ion also helps regulate acid-base balances by entering cells in response to rising carbon dioxide levels. As carbon dioxide increases, bicarbonate moves from the intracellular space to the extracellular space. In response, chloride tends to enter the cells.
Therefore, there are various circumstances where it is important to analyze serum and other bodily fluids to determine the amount of chloride ion. For example, hyperchloremia (high serum chloride) may indicate chronic hyperventilation, Cushing""s syndrome, dehydration, eclampsia, excess infusion of normal saline, kidney dysfunction, metabolic acidosis, or renal tubular acidosis. Hypochloremia (low serum chloride) may indicate Addison""s disease, burns, chronic respiratory acidosis (chronic hypoventilation), congestive heart failure, excessive sweating, gastric suction, over hydration, salt-losing nephritis, syndrome of inappropriate ADH secretion, or vomiting.
In response to this need, there have been developed various methods and devices to analyze bodily fluids and determine the amount of chloride ion in a sample. One early method was a calorimetric test of free chlorine. It detected the presence of the chloride ion by using a soluble silver salt and tolidine. However, this method lacked the precision required by most applications.
The need for more precise chloride ion measurements has led to the development of more accurate tests through a variety of methods. Most current methods for the determination of chloride ion are based on either electric or chemical methods. Electric methods include coulometric titration and the ion selective electrode method. The coulometric titration method is considered the most reliable method, if not the quickest. It can be performed with either manual or semi-automatic methods, but is difficult to perform in an automatic analytical system. The ion selective electrode method can have specificity problems and is prone to interference from proteins and surfactants.
The principal chemical method is colorimetry, wherein the concentration of the chloride ion is measured according to changes in color density. The colorimetry method is considered the most effective test for simple applications because it is less complicated than coulometric or ion selective electrode methods. The most common chloride determination colorimetry methods react mercuric thiocyanate and chloride ion to produce thiocyanate ion, which then forms a complex with ferric ion. The result is a characteristic red orange color, which deepens as the concentration of the chloride ion becomes higher, thus enabling colorimetry. Nevertheless, this method has drawbacks because both mercuric ions and the thiocyanate ions are harmful to the environment. As a result, each use of the reagents creates additional waste that requires costly care and treatment. Therefore, there is a need for calorimetric methods to determine chloride ion concentration that avoid the mercuric and thiocyanate ions.
U.S. Pat. No. 5,229,270 to Ono et al. (hereinafter xe2x80x9cOnoxe2x80x9d) discloses an environmentally safe quantitative assay and reagent for the determination of chloride ion in bodily fluids. Ono uses a reagent which contains deactivated xcex1-amylase, a compound capable of chelating calcium ion, a calcium chelate ion, a calcium chelate compound, and an xcex1-amylase measuring substance. The method comprises the steps of: (a) contacting a bodily fluid sample suspected of containing chloride ions with a reagent which comprises a compound capable of forming a chelate with a calcium ion, deactivated xcex1-amylase, a calcium chelate compound, and an xcex1-amylase activity-measuring substance; (b) determining the quantity of xcex1-amylase activity formed due to the presence of chloride ions in the bodily fluid sample, which is directly proportional to the amount of chloride ions present in the bodily fluid sample; and (c) determining the quantity of the chloride ions from the quantity of the xcex1-amylase activity by referring to a calibration curve. This method is capable of automation and has high ion specificity.
It has previously been shown that xcex1-amylase contains one chloride ion binding site per molecule. One early approach at describing the chloride ion effects on xcex1-amylase is described in Lifshitz, Ruth, Levitski, Alexander, Identity and Properties of the Chloride Effector Binding Site in Hog Pancreatic xcex1-Amylase, Biochemistry, Vol. 15, No. 9, 1976. (hereinafter, xe2x80x9cLifshitzxe2x80x9d). Lifshitz is specifically directed to determine the chloride ion binding site in xcex1-amylase. In reaching their conclusions, Lifshitz discusses various compounds and their effects upon the chloride ion binding site. For example, Lifshitz teaches that calcium-free xcex1-amylase is unable to bind chloride ion. Similarly, Lifshitz tested sodium fluoride (fluoride ion) and sodium acetate (acetate ion) to determine their effect upon the chloride ion affinity of deactivated xcex1-amylase. Lifshitz determined that neither fluoride ion nor sodium acetate had any appreciable effect upon the chloride ion affinity of deactivated xcex1-amylase. Lifshitz at 1990.
Notwithstanding the prior methods for assaying chloride ions using calcium-activated xcex1-amylase, there remains a continuing need for alternative systems for assaying chloride.
It is an object of the present invention to provide alternative methods, compositions, and assays to determine the quantity of chloride ion in biological samples.
It is another object of the present invention to provide alternative methods, compositions, and assays to determine the quantity of sodium ion in biological samples.
It is a further object of the invention to provide a chloride assay that is environmentally safe.
It is a yet a further object of the invention to provide a chloride assay that is simple to perform yet accurate.
In accordance with the present invention, there is provided a method for the determination of chloride ion in biological fluids. To perform clinical assays incorporating the discoveries of the present invention, one method for determining the concentration of chloride ions in samples comprises first preparing an enzyme reagent which includes xcex1-amylase that is substantially calcium-free and an xcex1-amylase activity detecting substrate. Next, the enzyme reagent, sodium ion, and a sample containing chloride ion to be assayed are combined, wherein the sodium ion is present in a higher concentration than the chloride ion. The quantity of xcex1-amylase activated due to the presence of sodium ions and chloride ions in the sample is then assayed. Finally, the quantity of the chloride ions is determined by reference to the assay of xcex1-amylase.
A composition for use in determining the concentration of chloride ions in bodily fluid samples comprises xcex1-amylase that is substantially calcium-free, an xcex1-amylase activity detecting substrate, and sodium ion, wherein the concentration of sodium ion is higher than that of the chloride ion to be assayed.
Yet another aspect of the invention comprises a method for determining the concentration of sodium ions in bodily fluid samples. The method comprises first preparing an enzyme reagent which includes xcex1-amylase that is substantially calcium-free and an xcex1-amylase activity detecting substrate. Next, the enzyme reagent, chloride ion, and a sample containing sodium ion to be assayed are combined, wherein the chloride ion is present in a higher concentration than the sodium ion. The quantity of xcex1-amylase formed due to the presence of sodium ions and chloride ions in the sample is then assayed. Finally, the quantity of the sodium ions is determined by reference to the assay of xcex1-amylase.
These and other objects and features of the present invention will become more fully apparent from the following description and appended claims, or may be learned by the practice of the invention as set forth hereinafter.